Viruses are widely distributed throughout the Kingdom Fungi, including fungi pathogenic to animals and plants. Fungal infections continue to have a significant impact on human health and well being. Similar to viruses of animals and plants, mycoviruses have potential utility for elucidating host biological processes and manipulating host phenotype. Progress in developing this potential is best illustrated by recent studies with members of the RNA virus family Hypoviridae. Hypoviruses persistently alter phenotypic traits, modulate gene expression and attenuate virulence of their fungal hosts. They are also the only symptomatic mycoviruses for which a reverse geneticssystem has been developed. Consequently, hypoviruses provide utility as biological control agents and as unique tools for identifying fungal virulence determinants and designing antimycotic therapeutic strategies. Substantial progress has been made in linking hypovirus-mediated regulation of specific host transcription factors with virus symptom expression and the regulated expression of a subset of virus-responsive genes. The hypovirus protease p48 was shown to be required for initiation but not for maintenance of viral RNA propagation. In a surprising series of results, host RNA silencing was shown to be required for production of viral defective RNAs and for the instability of recombinant viral RNA vectors. Three new Specific Aims have been designed to capture the full advantage of the hypovirus/C. parasitica experimental system for further development of these novel new findings: 1) characterize transient requirement of p48 for initiation but not for maintenance of viral RNA propagation, 2) further characterize hypovirus-responsive transcription factors and their contribution to hypovirus-mediated virulence attenuation and persistent symptom expression and 3) characterize the role of RNA silencing in defective viral RNA generation and recombinant viral vector instability. The results derived form completion of these coordinated specific aims will have immediate relevance to ongoing efforts to further enhance the utility of hypoviruses and other potentially efficacious RNA viruses for purposes of manipulating their hosts in novel and productive ways. The proposed research will also have broad relevance to three important areas of contemporary virology: viral RNA replication, the relationship between virus-mediated alterations of host gene expression and symptom expression and mechanisms underlying viral defective interfering RNA production and viral RNA vector instability. PUBLIC HEALTH RELEVANCE: Fungal infections continue to have a significant impact on human health and wellbeing. Virulence-attenuating fungal viruses, the hypoviruses, provide utility for identifying fungal virulence determinants and designing antimycotic therapeutic strategies. The proposed research will further enhance the utility of hypoviruses and other potentially efficacious RNA viruses for purposes of productively manipulating their host. The unique capabilities of the hypovirus experimental system will also be used to exploit novel new findings with broad relevance to viral RNA replication, the relationship between virus-mediated alterations of host gene expression and persistent symptom expression and the role of RNA silencing in viral defective interfering RNA production and viral RNA vector instability.